Evaluation of Drosophila for screening developmental toxicants: test results with eighteen chemicals and presentation of a new Drosophila bioassay.

The objective of this study was to generate a comprehensive data set of chemically induced malformations in Drosophila using a detailed morphological examination of the entire fly (phase one). These data were analyzed, in blind, with the goal of developing a standardized set of criteria which could be used in a new, rapid, and economical Drosophila bioassay useful in the preliminary screening for potential developmental toxicants. After 32 chemicals were tested, formalized criteria were developed to form the basis of a new Drosophila bioassay. These criteria were then applied to the data from the same 32 chemicals (phase two). The data from only 18 of these chemicals met all requirements for evaluation, e.g., statistical significance, minimum fly numbers, sufficient challenge concentration administered, etc. In the new bioassay, rather than the detailed and time-consuming examination of the entire fly for a multitude of morphological defects, only two specific anatomical sites are examined. These sites are the humeral bristle and the wing blade, with focus placed on two structural defects--a bent bristle and a notch in the wing. These defects were the only two external malformations among the multitude of defects observed in flies treated in the first phase with the 32 chemicals which demonstrated the following characteristics: 1) A consistent concentration-response in flies treated with a variety of developmental toxicants; 2) a lack of response with most presumptive non-developmental toxicants; and 3) consistently low-background incidences in control flies. In both phases, developing Drosophila were exposed to the test agents from the egg through three larval stages by incorporating a range of concentrations of each chemical into the culture medium. Emerging adults were examined for an array of defects as part of a detailed morphological examination in the first phase, including bent bristles and wing notches. In the second phase, only bent bristle and wing notch data were evaluated. The incidences of bent humeral bristles and wing notches from flies exposed to each of the 18 chemicals were compared with those of concurrent controls. Of the 18 chemicals that could be evaluated using the new bioassay, 13 were known developmental toxicants while the remaining 5 were presumptive negative agents. Ten of the 13 mammalian developmental toxicants were correctly identified with this test (false negative rate of 23%). Four of five apparent non-developmental toxicants were correctly identified for a false positive rate of 20%.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of 60 chemicals in a preliminary developmental toxicity test.

The number of chemicals in commerce which have not been evaluated for potential developmental toxicity is large. Because of the time and expense required by conventional developmental toxicity tests, an abbreviated assay is needed that will preliminarily evaluate otherwise untested chemicals to help prioritize them for conventional testing. A proposed short-term in vivo assay has been used in a series of studies in which a total of 60 chemicals were tested. Some were independently tested two or four times each. In this preliminary test, pregnant mice were dosed during mid-pregnancy and were then allowed to deliver litters. Litter size, birth weight, and neonatal growth and survival to postnatal day 3 were recorded as indices of potential developmental toxicity. Results in this assay and conventional mouse teratology tests were generally concordant. Conventional data were available for 14 chemicals (ten teratogens, one fetotoxin, three nonteratogens), of which 11 (nine teratogens, one fetotoxin, one nonteratogen) produced evidence of developmental toxicity. This included conventional data for three chemicals (ethylene glycol, diethylene glycol dimethyl ether, and triethylene glycol dimethyl ether) that were untested before the present study. As high priority candidates for conventional testing on the basis of results here, all were subsequently studied in a standard teratology assay and were confirmed to be teratogenic in mice. Additionally, one of them (ethylene glycol) plus a fourth high priority candidate for conventional study (diethylene glycol monomethyl ether) were subsequently tested in rats and were found to be teratogenic in that species.

Developmental toxicity of nine selected compounds following prenatal exposure in the mouse: naphthalene, p-nitrophenol, sodium selenite, dimethyl phthalate, ethylenethiourea, and four glycol ether derivatives.

Ethylene glycol dimethyl ether (EGdiME), diethylene glycol dimethyl ether (diEGdiME), triethylene glycol dimethyl ether (triEGdiME), diethylene glycol diethyl ether (diEGdiEE), ethylenethiourea (ETU), sodium selenite (SS), dimethyl phthalate (DMP), naphthalene (NAP), or p-nitrophenol (PNP) were administered by gavage for eight consecutive days to female CD-1 mice. Weight loss was insensitive as an index of sublethal adult toxicity and was inadequate for determining a maximum tolerated dose. LD50 values indicate that SS, NAP, and PNP were more toxic (8.4, 353.6, and 625.7 mg/kg, respectively) than the polyglycol ethers, ETU, and DMP (LD50 values ranged from 2525.8 to 6281.9 mg/kg). Each of the compounds was administered on d 7 through 14 to pregnant animals at a single dose estimated to be at or just below the threshold of adult lethality. In such a reproductive study, each of the compounds could be categorized on the basis of the pattern of maternal lethality and fetotoxicity which it produced. The number of dams with complete resorptions was significantly increased after administration of ETU, and no mice in the EGdiME-, diEGdiME-, or triEGdiME-treated groups delivered any viable offspring. Maternal lethality was significant in the EGdiME, triEGdiME, PNP, and NAP groups. There was a slight reduction in the average number of live pups per litter in the diEGdiEE- and PNP-treated groups and a significant reduction in the NAP group. The number dead per litter was increased with diEGdiEE. SS and DMP had no effect on maternal or fetal survival at the doses administered. Individual pup weight at d 1 postpartum was only significantly reduced by diEGdiEE, and no gross congenital abnormalities were detected in neonates from any treatment group. These results provide guidelines for the subsequent toxicity testing of these chemicals.

Results of testing fifteen glycol ethers in a short-term in vivo reproductive toxicity assay.

Fifteen glycol ethers were investigated for their potential to cause adverse reproductive toxic effects using an in vivo mouse screening bioassay. Pregnant mice were orally dosed once per day on days 7 through 14 of gestation at concentrations causing 0 to 41% maternal mortality. Reproductive endpoints included pup survival in utero (percent of live litters/pregnant survivors), pup perinatal and postnatal survival (number of live pups per litter, number of dead pups per litter, and pup survival to 2.5 days of age), and pup body weight statistics (weight at birth and weight at 2.5 days of age). The study was conducted in two phases: a dose range-finding phase using nonpregnant female mice, and a definitive reproductive phase using time-mated mice. The range-finding phase sought to identify, for each chemical, the maternal LD10 as the target dose. However, based upon reproductive phase results, such an exact dose was impractical to achieve. Thus, a range from the LD5 to the LD20 was considered a sufficient challenge dose that would not affect results due to high mortality, i.e., greater than the LD20. Glycol ethers were assigned to groups having different priorities for further testing based upon whether a sufficient challenge dose was administered and the degree of effects recorded for each chemical.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of propylene oxide for mutagenic activity in 3 in vivo test systems.

Propylene oxide (CAS No. 75-56-9) was tested for mutagenic activity following vapor exposure using 3 in vivo test systems. Rat dominant lethal and mouse sperm-head morphology assays were conducted using males exposed to propylene oxide at 300 ppm in a dynamic exposure chamber for 7 h per day on 5 consecutive days. A sex-linked recessive lethal test in Drosophila melanogaster employed a 24-h static exposure to propylene oxide at 645 ppm. Male mice were killed 1, 3, 5, 7, and 9 weeks post-exposure for evaluation of sperm-head morphology. Propylene oxide exposure did not result in an increase in abnormal forms. Male rats were mated with 2 virgin females per week for 6 weeks following exposure. A statistically significant increase in preimplantation losses and a statistically significant reduction in the number of living implants in the first post-exposure week did not appear to be treatment related. A highly significant increase in sex-linked recessive lethal mutations was observed in two germ cell stages (mature sperm and developing spermatocytes). These results warrant continued caution in potential human exposure to propylene oxide.

Drosophila as a tool for the rapid assessment of chemicals for teratogenicity.

Drosophila melanogaster is being investigated for its potential to aid in identifying priority chemicals for teratologic study. The method encompasses treating larvae over the entire metamorphosis period, i.e., from the egg through three instar stages to pupa formation, by incorporating the test chemical into the medium. Adult flies are systematically examined under a binocular microscope for external morphological anomalies. Data from treated flies can be compared with those from concurrent control flies using standard statistical tests. Results from this developmental work reveal a dramatic and reproducible response of Drosophila to various chemical treatments. Validation studies, testing known teratogens and nonteratogens, are necessary before such a system can be incorporated into existing teratologic screening regimens.

Acute toxicity of tetramethylbenzenes: durene, isodurene and prehnitene.

Oral LD50 (rat), primary skin irritation (rabbit), cutaneous sensitization (guinea pig) and eye irritation (rabbit) studies were conducted on the three tetramethylbenzene isomers: durene , isodurene and prehnitene. The order of oral toxicity was isodurene greater than prehnitene greater durene. Durene was not a skin irritant, while isodurene and prehnitene each produced a mild positive skin response (erythema). None of the tetramethylbenzenes were skin sensitizers or eye irritants. Durene, isodurene and prehnitene are only slightly toxic on an acute toxicologic basis and only pose an acute health hazard when injested in excessive quantities.